Methods for applying small volumes of reagents

ABSTRACT

Methods, devices and apparatus are disclosed for carrying out multiple chemical reactions, such as in situ synthesis of polynucleotides, on a surface comprising an array of discrete sites. Molecules are deposited at a predetermined number of the discrete sites on the surface for reaction at the discrete sites. The surface is positioned relative to an outlet of a fluid ejection device, which is activated to dispense a small volume of a fluid through the outlet to the surface to provide uniform coating of a continuous region of the surface comprising a multiple of the discrete sites. The fluid is dispensed as uniform particles having a diameter such that the uniform particles form a sheet to coat the continuous region of the surface. In one embodiment of the present invention, liquid is dispensed as uniform particles through a fluid ejection device activated by means of ultrasonic energy. The invention has particular application to the in situ synthesis of polynucleotides in arrays on a surface.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to the application of small volumes ofreagents to surfaces. In one aspect the invention relates to themanufacture of arrays formed and arranged by depositing compounds orsynthesizing large numbers of compounds on solid supports in apredetermined pattern. In another aspect this invention relates to thefield of bioscience in which arrays of oligonucleotide probes arefabricated or deposited on a surface and are used to identify or analyzeDNA sequences in cell matter. The present invention has a wide range ofapplication for synthesis and use of arrays of oligonucleotides orproteins for conducting cell study, for diagnosing disease, identifyinggene expression, monitoring drug response, determination of viral load,identifying genetic polymorphisms, and the like.

[0003] Significant morbidity and mortality are associated withinfectious diseases and genetically inherited disorders. More rapid andaccurate diagnostic methods are required for better monitoring andtreatment of these conditions. Molecular methods using DNA probes,nucleic acid hybridization and in vitro amplification techniques arepromising methods offering advantages to conventional methods used forpatient diagnoses.

[0004] Nucleic acid hybridization has been employed for investigatingthe identity and establishing the presence of nucleic acids.Hybridization is based on complementary base pairing. When complementarysingle stranded nucleic acids are incubated together, the complementarybase sequences pair to form double-stranded hybrid molecules. Theability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleicacid (RNA) to form a hydrogen bonded structure with a complementarynucleic acid sequence has been employed as an analytical tool inmolecular biology research. The availability of radioactive nucleosidetriphosphates of high specific activity and the development of methodsfor their incorporation into DNA and RNA has made it possible toidentify, isolate, and characterize various nucleic acid sequences ofbiological interest. Nucleic acid hybridization has great potential indiagnosing disease states associated with unique nucleic acid sequences.These unique nucleic acid sequences may result from genetic orenvironmental change in DNA by insertions, deletions, point mutations,or by acquiring foreign DNA or RNA by means of infection by bacteria,molds, fungi, and viruses.

[0005] The application of nucleic acid hybridization as a diagnostictool in clinical medicine is limited due to the cost and effortassociated with the development of sufficiently sensitive and specificmethods for detecting potentially low concentrations of disease-relatedDNA or RNA present in the complex mixture of nucleic acid sequencesfound in patient samples.

[0006] One method for detecting nucleic acids is to employ nucleic acidprobes that have sequences complementary to sequences in the targetnucleic acid. A nucleic acid probe may be, or may be capable of being,labeled with a reporter group or may be, or may be capable of becoming,bound to a support. Detection of signal depends upon the nature of thelabel or reporter group. Usually, the probe is comprised of naturalnucleotides such as ribonucleotides and deoxyribonucleotides and theirderivatives although unnatural nucleotide mimetics such as 2′-modifiednucleosides, peptide nucleic acids and oligomeric nucleosidephosphonates are also used. Commonly, binding of the probes to thetarget is detected by means of a label incorporated into the probe.Alternatively, the probe may be unlabeled and the target nucleic acidlabeled. Binding can be detected by separating the bound probe or targetfrom the free probe or target and detecting the label. In one approach,a sandwich is formed comprised of one probe, which may be labeled, thetarget and a probe that is or can become bound to a surface.Alternatively, binding can be detected by a change in thesignal-producing properties of the label upon binding, such as a changein the emission efficiency of a fluorescent or chemiluminescent label.This permits detection to be carried out without a separation step.Finally, binding can be detected by labeling the target, allowing thetarget to hybridize to a surface-bound probe, washing away the unboundtarget and detecting the labeled target that remains.

[0007] Direct detection of labeled target hybridized to surface-boundprobes is particularly advantageous if the surface contains a mosaic ofdifferent probes that are individually localized to discrete, knownareas of the surface. Such ordered arrays containing a large number ofoligonucleotide probes have been developed as tools for high throughputanalyses of genotype and gene expression. Oligonucleotides synthesizedon a solid support recognize uniquely complementary nucleic acids byhybridization, and arrays can be designed to define specific targetsequences, analyze gene expression patterns or identify specific allelicvariations.

[0008] In one approach, cell matter is lysed, to release its DNA asfragments, which are then separated out by electrophoresis or othermeans, and then tagged with a fluorescent or other label. The resultingDNA mix is exposed to an array of oligonucleotide probes, whereuponselective attachment to matching probe sites takes place. The array isthen washed and imaged so as to reveal for analysis and interpretationthe sites where attachment occurred.

[0009] In the preparation of arrays, reagents are applied topredetermined discrete locations on the surface of a substrate.Depending on the type of synthesis and array, the preparation mayinvolve application of reagents at discrete locations followed bytreatment of a portion or the entire surface with a different liquidreagent. The steps may be repeated a number of times sufficient toprepare the desired array. Examples of known methods for subjecting allor a portion of substrate surfaces to reagents include flooding, spincoating and flow cell assembly. Flooding the surface may be accomplishedby using, for example, a multi-nozzle piezoelectric pump head. Arelatively large volume of liquid is dispensed to contact the surfaceand assure that the dispensed reagents contact all of the desiredlocations. Spin coating is usually performed by dispensing the reagentat or near the center of the substrate followed by spinning to spreadthe reagent uniformly across the substrate.

[0010] The volume used to cover the substrate depends on the fluidproperty and the surface energy of the substrate. Some approaches usedfor in situ synthesis require a large relative volume to cover thesurface because small, dispensed volumes tend to cluster andnon-uniformly cover the surface. The reagent is then removed from thesubstrate within a high-speed spin step, which generates a considerableamount of waste. In the flow cell approach, a seal layer is brought incontact with the substrate at various support points (typically alongthe perimeter). A thin gap exists between the substrate and seal layer.By developing a pressure gradient across inlet and outlet channels,fluid can be forced to flow in the gap along the substrate. Althoughthis method can use considerably less volume than the flooding method orthe spin coat method, it has three major drawbacks. First, there is along fill time in order to support laminar flow. Second, it is prone toleaking if uniform pressure is not maintained. Third, variability insurface thickness will disturb the laminar flow resulting in air pocketsand hence non-uniform coverage.

[0011] 2. Description of the Related Art

[0012] U.S. Pat. No. 5,831,070 (Pease, et al.) discloses printingoligonucleotide arrays using deprotection agents solely in the vaporphase.

[0013] Sono-Tek Corporation brochure, copyright 1996.

SUMMARY OF THE INVENTION

[0014] One embodiment of the present invention is a method forconducting chemical reactions on a surface comprising an array ofdiscrete sites. Molecules are deposited at a predetermined number of thediscrete sites on the surface for reaction at the discrete sites. Thesurface is positioned relative to an outlet of a fluid ejection device,which is activated to dispense a small volume of a fluid through theoutlet to the surface to provide uniform coating of a continuous regionof the surface comprising a multiple of the discrete sites. The fluid isdispensed as uniform particles having a diameter such that the uniformparticles form a sheet to coat the continuous region of the surface.

[0015] Another embodiment of the present invention is a method forforming an array of molecules at discrete sites on a surface. Moleculeprecursors are applied to predetermined discrete sites on the surface. Asmall volume of a liquid is dispensed to uniformly coat a continuousregion of the surface comprising a multiple of the discrete sites. Thesmall volume of liquid is dispensed as uniform particles through a fluidejection device activated by means of ultrasonic energy.

[0016] Another embodiment of the present invention is a method forforming an array of polynucleotides at discrete sites on a surface.Reagents are applied to predetermined discrete sites on the surface. Thereagents are selected from the group consisting of nucleotides andpolynucleotides. A volume of a liquid is dispensed to uniformly coat thesurface with liquid. The volume of liquid is dispensed as particles ofuniform diameter through a fluid ejection device activated by means ofultrasonic energy. Step (a) or step (b) may optionally be repeated.

[0017] Another embodiment of the present invention is a method forforming an array of polynucleotides at discrete sites on a surface.Nucleotide reagents are applied to predetermined discrete sites on thesurface. A volume of a liquid of about 1 nanoliter to about 1000nanoliters is dispensed to uniformly coat the surface with liquid. Theliquid is dispensed as particles of uniform diameter of about 1 micronsto about 500 microns through a fluid ejection device activated by meansof ultrasonic energy at a frequency of about 5 kilohertz to about 300kilohertz. The liquids comprise agents selected from the groupconsisting of wash liquids, deblocking agents and deprotection agents.Step (a) or step (b) optionally may be repeated.

[0018] Another embodiment of the present invention is an apparatus forforming an array of polynucleotides at discrete sites on a surface. Theapparatus comprises a device for dispensing reagents to predetermineddiscrete sites on said surface and a fluid ejection device activated bymeans of ultrasonic energy. The fluid ejection device dispenses a volumeof a liquid as particles of uniform diameter to uniformly coat thesurface with liquid. The reagents are selected from the group consistingof nucleotides and polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a section diagram depicting a device in accordance withthe present invention.

[0020]FIG. 2 is a schematic diagram depicting an apparatus in accordancewith the present invention.

DEFINITIONS

[0021] Before proceeding further with a description of the specificembodiments of the present invention, a number of terms will be defined.

[0022] Polynucleotide—a compound or composition that is a polymericnucleotide or nucleic acid polymer. The polynucleotide may be a naturalcompound or a synthetic compound. In the context of an assay, thepolynucleotide is often referred to as a polynucleotide analyte. Thepolynucleotide can have from about 2 to 5,000,000 or more nucleotides.The larger polynucleotides are generally found in the natural state. Inan isolated state the polynucleotide can have about 2 to 50,000 or morenucleotides, usually about 10 to 20,000 nucleotides, more frequently 100to 10,000 nucleotides. It is thus obvious that isolation of apolynucleotide from the natural state often results in fragmentation.The polynucleotides include nucleic acids, and fragments thereof, fromany source in purified or unpurified form including DNA (dsDNA andssDNA) and RNA, including tRNA, mRNA, rRNA, mitochondrial DNA and RNA,chloroplast DNA and RNA, DNA/RNA hybrids, or mixtures thereof, genes,chromosomes, plasmids, the genomes of biological material such asmicroorganisms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi,plants, animals, humans, and the like. The polynucleotide can be only aminor fraction of a complex mixture such as a biological sample. Alsoincluded are genes, such as hemoglobin gene for sickle-cell anemia,cystic fibrosis gene, oncogenes, cDNA, compounds produced synthetically,e.g., PNA as described in U.S. Pat. No. 5,948,902 and references citedtherein, which can hybridize with naturally occurring nucleic acids in asequence specific manner analogous to that of two naturally occurringnucleic acids, and the like.

[0023] The polynucleotide can be obtained from various biologicalmaterials by procedures well known in the art. The polynucleotide, whereappropriate, may be cleaved to obtain a fragment that contains a targetnucleotide sequence, for example, by shearing or by treatment with arestriction endonuclease or other site specific chemical cleavagemethod.

[0024] The polynucleotide, or a cleaved fragment obtained from thepolynucleotide, will usually be at least partially denatured or singlestranded or treated to render it denatured or single stranded. Suchtreatments are well known in the art and include, for instance, heat oralkali treatment, or enzymatic digestion of one strand. For example,dsDNA can be heated at 90 to 100° C. for a period of about 1 to 10minutes to produce denatured material.

[0025] Oligonucleotide—a polynucleotide, usually single stranded,usually a synthetic polynucleotide but may be a naturally occurringpolynucleotide. The oligonucleotide(s) are usually comprised of asequence of at least 5 nucleotides, preferably, 10 to 100 nucleotides,more preferably, 20 to 50 nucleotides, and usually 10 to 30 nucleotides,more preferably, 15 to 30 nucleotides. The oligonucleotides includeoligonucleotide probes and oligonucleotide primers.

[0026] Methods of oligonucleotide synthesis include phosphotriester andphosphodiester methods (Narang, et al. (1979) Meth. Enzymol 68:90) andsynthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters22:1859-1862) as well as phosphoramidite techniques (Caruthers, M. H.,et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988)) andothers described in “Synthesis and Applications of DNA and RNA,” S. A.Narang, editor, Academic Press, New York, 1987, and the referencescontained therein.

[0027] Nucleoside triphosphates—nucleosides having a 5′-triphosphatesubstituent. The nucleosides are pentose sugar derivatives ofnitrogenous bases of either purine or pyrimidine derivation, covalentlybonded to the 1′-carbon of the pentose sugar, which is usually adeoxyribose or a ribose. The purine bases include adenine (A), guanine(G), inosine (I), and derivatives and analogs thereof. The pyrimidinebases include cytosine (C), thymine (T), uracil (U), and derivatives andanalogs thereof. Nucleoside triphosphates include deoxyribonucleosidetriphosphates such as the four common deoxyribonucleoside triphosphatesdATP, dCTP, dGTP and dTTP and ribonucleoside triphosphates such as thefour common triphosphates rATP, rCTP, RGTP and rUTP. The term“nucleoside triphosphates” also includes derivatives and analogsthereof, which are exemplified by those derivatives that are recognizedand polymerized in a similar manner to the underivatized nucleosidetriphosphates.

[0028] Nucleotide—a base-sugar-phosphate combination that is themonomeric unit of nucleic acid polymers, i.e., DNA and RNA. The term“nucleotide” as used herein includes modified nucleotides, which a unitthat contains a modified base, sugar or phosphate group.

[0029] DNA—deoxyribonucleic acid.

[0030] RNA—ribonucleic acid.

[0031] cDNA—a DNA copy of a corresponding RNA. It can be a sequence ofDNA obtained by reverse transcription of an RNA molecule. It can includedouble-stranded or single stranded DNA obtained by amplification. Anexample, by way of illustration and not limitation, is thedouble-stranded DNA product obtained by PCR amplification of a bacterialplasmid insert. The DNA sequence inserted in the plasmid is previouslyobtained from reverse transcription of the corresponding RNA.

[0032] Nucleoside—is a base-sugar combination or a nucleotide lacking aphosphate moiety.

[0033] The term “support” or “substrate” refers to a porous ornon-porous water insoluble material. The term “surface” refers to asurface or outer side of a support or substrate; the surface depends onthe particular shape of the support or substrate. The support can haveany one of a number of shapes, such as square, circular, rectangular,spherical, and the like such as found in a strip, plate, disk, and soforth. The support can be hydrophilic or hydrophobic or capable of beingrendered hydrophilic or hydrophobic. Such supports include naturalpolymeric materials, particularly cellulosic materials and materialsderived from cellulose, such as fiber containing papers, e.g., filterpaper, chromatographic paper, etc.; synthetic or modified naturallyoccurring polymers, such as nitrocellulose, cellulose acetate, poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc., either used by themselves or in conjunctionwith other materials; flat glass whose surface has been chemicallyactivated to support binding or synthesis of polynucleotides; glassavailable as Bioglass; other types of silicon based supports; ceramics;metals, and the like. The surface of a support may be renderedhydrophobic by treatment with a reagent such as a silane, e.g.,fluoroalkylsilane, and the like. Binding of oligonucleotides to asupport or surface may be accomplished by well-known techniques,commonly available in the literature. See, for example, A. C. Pease, etal., Proc. Nat. Acad. Sci. USA 91:5022-5026 (1994). Other approaches arediscussed briefly herein.

[0034] Monomer—a chemical entity that can be covalently linked to one ormore other such entities to form an oligomer or polymer. Examples ofmonomers include nucleotides, modified nucleotides, amino acids, iminoacids, saccharides, peptoids, and the like. In general, the monomersused in conjunction with the present invention have first and secondsites (e.g., C-termini and N-termini, or 5′and 3′sites) suitable forbinding of other like monomers by means of standard chemical reactions(e.g., condensation, nucleophilic displacement of a leaving group, orthe like), and a diverse element that distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid sidechain, a nucleotide base, etc.). The initial substrate-bound monomer isgenerally used as a building block in a multi-step synthesis procedureto form a complete ligand usually in a desired sequence, such as in thesynthesis of oligonucleotides, oligopeptides and the like.

[0035] Oligomer—a chemical entity that contains a plurality of monomers.As used herein the terms “oligomer” and “polymer” are usedinterchangeably as it is generally although not necessarily smaller“polymers” that are prepared or attached using the functionalizedsubstrates of the present invention. Example oligomers and polymersinclude polydeoxyribonucleotides, polyribonucleotides, otherpolynucleotides that are C-glycosides of a purine or pyrimidine base, orother modified polynucleotides, polypeptides, polysaccharides, and otherchemical entities that contain repeating units of like chemicalstructure. In the practice of the present invention, oligomers generallycomprise about 6 to about 20,000 monomers, preferably, about 10 to about10,000, more preferably about 15 to about 4,000 monomers.

[0036] Amino acid—includes not only the L-, D- and non-chiral forms ofnaturally occurring amino acids (alanine, arginine, etc.) but alsomodified amino acids, amino acid analogs, and other chemical compoundsthat can be incorporated in conventional oligopeptide synthesis, e.g.,4-nitrophenylalanine, isoglutamic acid, isoglutamine,ε-nicotinoyllysine, isonipecotic acid, tetrahydroisoquinoleic acid,α-aminoisobutyric acid, sarcosine, citrulline, cysteic acid,t-butylglycine, t-butyl alanine, phenylglycine, cyclohexylalanine,β-alanine, 4-aminobutyric acid and the like.

[0037] Blocking and deblocking—relate to the addition and removal ofchemical blocking groups using conventional materials and techniqueswithin the skill of the art and/or described in the pertinentliterature. Blocking agents are those agents that are bound to a monomerunit and which may be selectively removed therefrom to expose an activesite. The blocking may be, for example, a dimethoxytrityl group and thelike linked to a nucleotide by a 5′-hydroxyl position as used inpolynucleotide synthesis. The blocking group may be, for example, anamine group and the like linked to an amino acid as used in thesynthesis of peptides.

[0038] Protection and deprotection—relate to the addition and removal ofchemical protecting groups using conventional materials and techniqueswithin the skill of the art and/or described in the pertinentliterature; for example, reference can be made to Greene, et al.,Protective Groups in Organic Synthesis, 2^(nd) Ed., New York, John Wiley& Sons (1991). Protecting groups prevent the site to which they areattached from participating in the chemical reaction to be carried out.Usually, the protecting groups may be selectively removed to expose anactive site.

DETAILED DESCRIPTION OF THE INVENTION

[0039] In its broadest application the present invention is directed tomethods for conducting chemical reactions on a surface of a substrate atdiscrete sites comprising an array. The chemical reactions may beconcerned, for example, with the synthesis of molecules on the surfaceof the array or with carrying out a diagnostic procedure utilizing thearray. In carrying out such chemical reactions one or more steps mayinvolve delivering a volume of fluid to a surface to provide uniformcoating of a continuous region of the surface comprising a multiple ofthe discrete sites. In these steps a fluid ejection device is employedthat is activated to dispense a small volume of fluid through an outletto the surface. The present invention is applicable to situations inwhich liquids have previously been dispensed by known methods to asurface by flooding the surface or a portion thereof with liquid. In thepresent invention fluid is dispensed as uniform particles having adiameter such that the uniform particles form a thin sheet to coat thedesired region of the surface.

[0040] A number of advantages over the known methods may be realized inthe practice of the present invention. The present method produces athinner layer of liquid deposited on a surface than that produced byprior methods such as flow cell assembly. Such an advantage isparticularly important for surfaces that are hydrophobic because itavoids the beading and non-uniform coverage that results when largerquantities of liquid are applied to the surface. Uniform coverage of ahydrophobic surface is achievable with the present invention because thesmall volume of liquid dispensed tends to form a layer on the surfacerather than form beads. Liquid movement to hydrophilc sites on thesurface is promoted. Another advantage of the present invention is thatliquid accumulates at the discrete sites on the surface in a more facilemanner than in methods known in the art. This surprising result furtherfacilitates bringing reagents to the desired locations for reaction atthe discrete sites. Such an advantage is particularly important in thepresent invention in which very small volumes of liquid are dispensed tothe surface. A further advantage of the present invention is thatmultiple thin layers of liquid may be applied to the surface in aparticular step. As a result diffusion of reagents is greatly reduced.This is important because fresh reagents may be supplied to the sites ofreaction by applying successive 2 to 5 separate thin layers of reagentsover a short period of time in accordance with the present invention. Inknown methods such as flow cell assembly techniques the layers of liquidare much thicker. After reaction of reagents at the site, additionalreactants from the liquid must diffuse to the reaction site.

[0041] The present invention has application generally to conductingchemical reactions on a surface of a support or a substrate. The presentinvention is described herein for purposes of illustration primarilywith regard to the synthesis of arrays of oligonucleotides. However, theinvention has application to the preparation of other molecules as wellas to other types of manipulations involving chemical reactions. Thetypes of chemical reactions that may be carried out using the presentinvention include, by way of illustration and not limitation, synthesisof polymeric materials such as biomolecules, e.g., polynucleotidesincluding oligonucleotides and proteins including peptides, polyalcoholssuch as polysaccharides, e.g., carbohydrates, oligosaccharides, and thelike; conjugation of molecules such as the conjugation of reportergroups or labels to nucleic acids or nucleotides, proteins such asenzymes, antibodies, and the like; diagnostic procedures such as assaysinvolving ligands and receptors such as antibody-antigen orantibody-hapten binding, nucleic acid hybridization, and so forth;molecular biological reactions such as those involving enzymes, e.g.,amplification procedures such as polymerase chain reaction, ligase chainreaction, restriction enzyme reactions; and so forth. The presentinvention has particular application to chemical reactions involvingmultiple steps and a large number of compounds such as in the synthesisof combinatorial libraries and polynucleotide and peptide arrays.

[0042] The methods and reagents of the present invention areparticularly useful in the area of the preparation of oligonucleotidearrays and, in particular, the preparation of such arrays by in situsynthesis. In the field of bioscience, arrays of oligonucleotide probes,fabricated or deposited on a surface, are used to identify DNA sequencesin cell matter. The arrays generally involve a surface containing amosaic of different oligonucleotides or sample nucleic acid sequencesthat are individually localized to discrete, known areas of the surface.In one approach, multiple identical arrays across a complete frontsurface of a single substrate are used. However, the arrays produced ona given substrate need not be identical and some or all could bedifferent. Each array may contain multiple spots or features and eacharray may be separated by spaces. A typical array may contain from 100to 100,000 or more features. Each oligonucleotide on the array has alength typically in the range of about 10 to about 100 base pairs. Allof the features may be different, or some or all may be the same. Eachfeature may carry a predetermined polynucleotide having a particularsequence or a predetermined mixture of polynucleotides. While arrays maybe separated from one another by spaces, and the features may beseparated from one another by spaces, such spaces in either instance arenot essential.

[0043] The size of the array may be varied depending on the applicationas discussed herein. Fewer or more discrete sites may be employed,depending on the nature of the chemical reactions involved, costconsiderations, and so forth. The spacing between sites on the device isdetermined by the ease of fabrication, the requirement for resolutionbetween the various sites, and the number of sites desired on a device.However, particular spacing between sites or special arrangement orgeometry of the sites is not necessary for device function. Anycombination of micro-locations can operate over the complete area of thesurface. As mentioned above, molecules such as specific bindingmolecules, chemical and analytical reagents, and the like may beattached to the surface.

[0044] Ordered arrays containing a large number of oligonucleotides havebeen developed as tools for high throughput analyses of genotype andgene expression. Oligonucleotides synthesized on a solid supportrecognize uniquely complementary nucleic acids by hybridization, andarrays can be designed to define specific target sequences, analyze geneexpression patterns or identify specific allelic variations. The arraysmay be used for conducting cell study, for diagnosing disease,identifying gene expression, monitoring drug response, determination ofviral load, identifying genetic polymorphisms, analyze gene expressionpatterns or identify specific allelic variations, and the like.

[0045] Various ways may be employed to produce an array ofpolynucleotides on supports or surfaces such as glass, metal, plasticand the like. Such methods are known in the art. One such method isdiscussed in U.S. Pat. No. 5,744,305 (Fodor, et al.) and involves solidphase chemistry, photolabile protecting groups and photolithography.Binary masking techniques are employed in one embodiment of the above.In another approach ink jet technology may be used to spotpolynucleotides and other reagents on a surface as described, forexample, in PCT application WO 89/10977. Other methods include thosedisclosed by Gamble, et al., WO97/44134; Gamble, et al., WO98/10858;Baldeschwieler, et al., WO95/25116; Brown, et al., U.S. Pat. No.5,807,522; and the like.

[0046] In the above approaches to forming arrays using in situsynthesis, the chemistry involved may include monomers that arenucleoside triphosphates used to form the polynucleotides usually byphosphate coupling, either direct phosphate coupling or coupling using aphosphate precursor such as a phosphite coupling. Such coupling thusincludes the use of amidite (phosphoramidite), phosphodiester,phosphotriester, H-phosphonate, phosphite halide, and the like coupling.One preferred coupling method is the phosphoramidite coupling, which isa phosphite coupling. In using this coupling method, after the phosphitecoupling is complete, the resulting phosphite is oxidized to aphosphate. Oxidation can be effected with oxygen to give phosphates orwith sulfur to give phosphorothioates. The phosphoramidites aredissolved in anhydrous acetonitrile to give a solution having a givenratio of amidite concentrations. The mixture of known chemicallycompatible monomers is reacted to a solid support, or further along, maybe reacted to a growing chain of monomer units. For a more detaileddiscussion of the chemistry involved in the above synthetic approaches,see, for example, U.S. Pat. No. 5,436,327 at column 2, line 34, tocolumn 4, line 36, which is incorporated herein by reference in itsentirety.

[0047] As seen from the above discussion, arrays may be fabricated insitu, adding one base pair at a time to a primer site. Affymetrix, forexample, uses photolithography to uncover sites, which are then exposedand reacted with one of the four base pair phosphoramidites. Inphotolithography the surface is first coated with a light-sensitiveresist, exposed through a mask and the pattern is revealed by dissolvingaway the exposed or the unexposed resist and, subsequently, a surfacelayer. A separate mask must be made for each pattern, which may involvefour patterns for each base pair in the length of the probe. Muchoverhead is involved in preparing the masks for photolithography, whichmay number 80 for probes of length 20, thus rendering this techniquebest suited for very high volume production. There are also problems incontrolling the etching reaction and in registering masks between eachstep.

[0048] Another in situ method employs inkjet printing technology todispense the appropriate phosphoramidite onto the individual probesites. For example, see U.S. Pat. No. 5,700,637 and PCT WO 95/25116.Another method involves electrochemically patterning a surface. Anelectrolyte overlying the surface and an array of electrodes adjacent tothe surface and in contact with the electrolyte is provided. Thepotential of one or more electrodes of the array is altered so as todeposit or remove or chemically modify a substance on the surfaceadjacent the electrode. Several such treatments may be performed insequence using different electrodes of the array. The method may be usedfor step-wise chemical synthesis of, for example, oligonucleotidestethered to the surface.

[0049] In a similar approach a self-addressable, self-assemblingmicroelectronic device is used to carry out and control multi-step andmultiplex molecular biological reactions, such as biopolymer synthesis,nucleic acid hybridization, antibody-antigen reaction, and diagnostics,in microscopic formats. The device electronically can control thetransport and attachment of specific binding entities and otherreactants to specific microlocations.

[0050] Array plates have been disclosed where a glass support surface iscoated with a positive or negative photoresist substance and thenexposed to light and developed to create a patterned region of a firstexposed surface and a photoresist coated surface on the support. Thefirst exposed surface is reacted with a fluoroalkylsilane to form astable fluoroalkylsiloxane hydrophobic matrix on the first exposedsurface. The photoresist coat on the surface is removed so as to form asecond exposed surface, which is reacted with a hydroxy- oraminoalkylsilane so as to convert the second exposed surface to aderivatized hydrophilic binding site region and thus form the arrayplate.

[0051] Many other methods have been put forth for fabricating sucharrays. In one approach oligonucleotide probes are spotted on a suitablesurface to produce an array. For this purpose, pre-synthesized probesare employed. In another approach a substrate is prepared upon which islocated microdrop-sized loci at which chemical compounds are synthesizedor diagnostic tests are conducted. The loci are formed by applyingmicrodrops from which a microdrop is pulse-fed onto the surface of thesubstrate.

[0052] In other disclosures U.S. Pat. No. 5,474,796 (Brennan) disclosesa method for making array plates. U.S. Pat. No. 5,445,934 (Fodor, etal.) discusses an array of oligonucleotides on a solid substrate.

[0053] The present invention has application to the aforementionedmethods for fabricating arrays. In one aspect the invention concerns amethod for forming an array of molecules at discrete sites on a surface.Molecule precursors are applied to the surface at discrete sites. Themolecule precursors may be monomers such as, for example, amino acids,nucleotides, saccharides, peptoids, and the like, or polymers includingpolysaccharides, polymers having drugs linked to a polymeric backbone,biopolymers such as poly (amino acids) such as peptides and proteins,oligonucleotides, polynucleotides, and the like. Accordingly, theinvention herein has application to both in situ synthesis as well asthe synthesis of molecules by attachment of whole molecules to asurface. In either of the above synthetic approaches, the methods mayinclude one or more steps involving contacting the surface comprisingthe discrete sites with solutions of monomers or polymers which may alsocontain activators such as tetrazole, DCI and the like; solutions ofcoupling reagents such as, e.g., phosphoramidites such as cyanoethylphosphoramidite nucleotides; solutions of capping reagents to truncateunreacted nucleosides from further participation in subsequent monomeraddition cycles such as, e.g., acetic anhydride and 1-methylimidazole toacetylate free 5′-hydroxyl groups; wash solutions such as organicsolvents or buffers to remove unreacted reagents; solutions of chemicalreactants such as blocking and deblocking agents such as proticsolvents, trichloroacetic acid, dichloroacetic acid and the like;protecting and deprotecting reagents; acidic solutions such as, e.g.,solutions of acids for removal of dimethoxytrityl groups by acidhydrolysis; basic solutions; solutions of oxidizing agents such as,e.g., iodine in tetrahydrofuran/water/pyridine and the like; solutionsof reducing agents; solutions of carrier materials; and so forth. Thepresent invention may be employed to dispense liquids in all of theabove circumstances.

[0054] The attachment chemistry for carrying out the known syntheticmethods is sometimes referred to a “priming” the surface. To this end,the surface is modified so as to prepare the surface for attachment ofthe monomeric building blocks. This surface may be the surface itself oran overcoat layer. The surface may be modified with groups or couplingagents to covalently link the initial nucleoside to the surface.Representative groups include, by way of illustration and notlimitation, amino, especially primary amino, hydroxyl, thiol, sulfonicacid, phosphorous and phosphoric acid, particularly in the form of acidhalides, especially chloride and bromide, and carboxyl, and the like.The reactive groups are conveniently attached to the surface commonlythrough a hydrocarbyl radical such as an alkylene or phenylene divalentradical.

[0055] In one embodiment, the present invention has application to thedeblocking steps often utilized in oligonucleotide synthesis whereinthere may be several sites on a nucleoside, for example, of similarchemical nature, e.g., hydroxyl groups. The synthesis may involveblocking certain sites from reaction with protecting groups. Nucleosidereagents may be used that comprise the blocking group. As explainedabove, a blocking group is one that is chemically bound to a monomerunit and which may be removed. The blocking group is attachedtemporarily to a potentially reactive site so as to prevent it fromreacting. The blocking group assists in avoiding unwanted sidereactions. The blocking groups are generally stable during the reactionsinvolved and yet removable to yield the original site. The presentinvention may also be used in the latter stages of the synthesis todispense deprotecting agents for removal of protecting groups.

[0056] Phosphoramidite chemistry and solid phase oligonucleotidesynthesis protocols often use a blocking group such as a dimethoxytritylprotecting group for the 5′-hydroxyl of nucleosides. A phosphoramiditefunctionality is utilized at the 3′-hydroxyl position. Phosphoramiditesynthesis generally proceeds form the 3′to the 5′of the ribose ordeoxyribose sugar component of he phosphoramidite nucleoside. The 5′endof the growing chain is coupled with the 3′ phosphoramidite of theincoming base to form a phosphite triester intermediate. The 5′-hydroxylof the added base is often blocked by a blocking group so only one newbase is added to the growing chain at a time. Any unreacted 5′-hydroxylgroups are capped off to stop the synthesis of this chain, which wouldbe one base short at the end of the synthesis. The triester intermediateis subjected to iodine oxidation after each coupling reaction to yield amore stable phosphotriester intermediate. Without oxidation, theunstable phosphite triester linkage cleaves under the acidic conditionsof subsequent synthesis steps.

[0057] Attachment and removal of the blocking groups generally iseffected globally by one of the methods mentioned above such as floodingthe surface, spin coating and flow cell assembly. In accordance with oneaspect of the present invention, a continuous region of the surfacecomprising a multiple of discrete sites is exposed to a solutioncomprising reagents for conducting the deblocking step.

[0058] By the term “discrete sites” is meant a specific region, e.g.,spot, point or the like, on a surface that contains a feature such as amolecule precursor, molecule, and so forth. The discrete sites may beisolated or non-isolated, shielded or unshielded, continuous ordiscontinuous, connected or unconnected. The discrete sites may beestablished by etching, barrier formation, masking, and the like or bydepositing reagents on a surface. By the term “continuous regioncomprising a multiple of discrete sites” is meant a portion of, or theentire surface, comprising the active discrete sites as distinguishedfrom the discrete sites themselves. Usually, the solution comprisingsuch reagents is contacted with the entire surface comprising thediscrete sites.

[0059] Usually, the present methods involve positioning the surfacerelative to the outlet of a fluid ejection device. For example, thesurface may be mounted on a linear stage and moved in position relativeto the fluid ejection device above the stage. In another approach thesurface may be rotated to the fluid ejection device, which is movedradially relative to the surface. Other ways of positioning the surfacerelative to the fluid ejection device include a combination of the aboveapproaches.

[0060] The nature of the fluid ejection device is dependent on the typeof energy used to activate the device. In general, the fluid ejectiondevice should be capable of dispensing a small volume of fluid throughan outlet to provide uniform coating of the continuous region of thesurface. To provide such uniform coating the fluid should be dispensedas particles of substantially uniform size. The term “substantiallyuniform size” means that the diameter of each of the particles does notvary more than about 50%, usually, not more than about 25% and desirablynot more than about 5 to about 0%. The variation in the diameter of theparticles can be tolerated to a greater degree where the particles arewithin the average diameter range set forth below. The average diameterof the particles is generally about 1 to about 200 microns, usually,about 10 to about 150 microns, more usually, about 15 to about 100microns. Preferably, the particles of fluid are dispensed as a mist. Thediameter of the outlet should be larger than the diameter of theparticles to avoid clogging of the outlet. The diameter of the outlet,therefore, should be about 10 to about 250 times as large, usually about40 to about 150 times as large, as the average diameter of theparticles. In general, the diameter of the outlet is about 50 microns toabout 250 millimeters, usually, about 150 to about 25 millimeters.

[0061] One consideration for the dimensions of the nozzle relates to theshape of the spray desired. The spray may be conical, flat, narrow suchas in a microspray, and the like. For conical spray the diameter of theoutlet usually falls in the upper part of the ranges set forth above forthe diameter of the outlet in general. For flat spray the diameter ofthe outlet usually falls in the middle of the above range and formicrospray the diameter of the outlet usually falls in the lower part ofthe above range. Another consideration in the dimensions of the outletand in the energy levels used for dispensing the liquid is the viscosityof the liquid. In general, the higher the viscosity of the liquid, thegreater the energy levels and the greater the dimensions of the outlet.The viscosity of the liquid should allow the liquid to be dispensed asuniform particles as discussed above. Accordingly, the viscosity of theliquid is generally about 0.1 to about 10 centipoise, usually about 0.5to about 2.5 centipoise. The fluid ejection device is usually a nozzlethat comprises a nozzle stem having an outlet. Other types of outletsand configurations may be employed. Compressed air may be employed toassist in focusing the liquid exiting the outlet. Usually, thecompresses air is dispensed through a channel adjacent the outlet. Thepressure employed may be about 0.1 to about 5 pounds per square inch.

[0062] The volume of liquid dispensed is usually about 1 to about 1000nanoliters per square centimeter of surface, more usually, about 20 toabout 100 nanoliters per square centimeter. In general, the volume ofliquid is a minimal volume to provide uniform coating of the surface.The layer of coating of liquid provided in the present invention isabout 5 to about 1000 microns, usually about 20 to about 500 microns,more usually, about 25 to about 125 microns. The primary concern incoating the surface uniformly is that the necessary amount of reagentsis delivered to the discrete sites within the continuous region that isexposed to the liquid. One wishes to deliver reagents such asdeprotection reagents to all of the desired sites so that the reagentmay accomplish its expected purpose. The prior art approaches accomplishsuch delivery by exposing the surface to a considerable excess volume ofthe liquid containing the reagents. We have found unexpectedly andagainst the wisdom in the art that coating the surface uniformly withliquid reagents using a minimal amount of liquid dispensed inparticulate form as described above successfully accomplishes the abovepurpose.

[0063] The liquid is dispensed in about 0.1 to about 10 seconds, usuallyabout 0.5 to about 5 seconds. It is an advantage of the presentinvention that a short dispense time may be utilized compared to some ofthe techniques of the prior art. The flow rate for liquid into thenozzle of a suitable device should be about 10 microliters to about 5000microliters per second, usually about 200 to about 3000 microliters persecond. In general, the range for flow rate is large for ultrasonicactivation since atomization relies only on liquid being introduced ontothe atomizing surface and not on pressure.

[0064] The nozzle of a fluid ejection device may be fabricated from anymaterial that is compatible, usually substantially non-reactive, withthe liquid and reagents to be dispensed and with the particular means ofactivation employed for the device. The materials include, by way ofexample and not limitation, metal such as stainless steel, titanium,platinum, etc., plastic, ceramic, Teflon®-coated materials and the like.Various parts of the nozzle may be fabricated from different materialsdepending on the function of the part.

[0065] The fluid ejection device may be activated by means of ultrasonicenergy, mechanical energy, electrical energy, thermal energy and thelike. Examples of fluid ejection devices that are activated bymechanical, thermal or electrical energy include, e.g., inkjet devicesand the like.

[0066] Preferably, the fluid ejection device is activated by ultrasonicenergy. An ultrasonic dispense head such as a nozzle may be used toproduce a fine mist of liquid reagent over a substrate. By moving thenozzle relative to the substrate, the surface can be coated with aminimal volume of liquid (thin mist layer). The coating process is fastand free of mechanical obstructions. High frequency sound waves areemployed using ultrasonic nozzles to produce atomization. The ultrasonicenergy typically has a frequency of about 5 to about 300 kHz, usually,about 25 to about 150 kHz. For ultrasonic atomization power levels aregenerally from about 0.1 to about 50 watts, usually about 1 to about 15watts. It should be noted that the power levels are generally chosen tomaximize efficiency. Any ultrasonic device that is capable of producinga spray of liquid having the characteristics discussed above may beemployed in the present method.

[0067]FIG. 1 depicts a fluid ejection device in accordance with thepresent invention. The device 10 comprises housing 12 having fluid inlet14, which typically is in fluid communication with a reservoir (notshown). Housing 12 also has an input orifice 16 that is in communicationwith an ultrasonic generator (also not shown). Air inlet 18, which is incommunication with a source of compressed air (also not shown), is alsopresent in housing 12. Fluid inlet 14 communicates with nozzle body 20at one end thereof. At the other end of 20 is droplet outlet 22, whichis in communication with inlet 14 by means of channel 24. Housing 12also has air outlet 26 adjacent droplet outlet 22. Nozzle body 20comprises piezo transducers 28, which are coupled to body 20.

[0068] A particular device for accomplishing the purposes of the presentinvention is one in which high frequency electrical energy from anultrasonic generator is received by a transducer such as a piezoelectrictransducer, which converts such energy into vibratory mechanical motionat the same frequency. Amplifiers may be coupled to the transducers toamplify the motion. In one approach amplification may be realized by astep transition from a large to a small diameter of a front horn of thenozzle. The excitation created by the transducers produces standingwaves along the length of the nozzle. The amplitude of the wave ismaximized at the atomizing surface, which is usually located at the endof a small diameter portion of the nozzle. Compressed air at a pressureof about 0.5 to about 1.5 psi, typically about 1 psi is introduced intoan ultrasonic device, usually into a diffusion chamber of an air shroudof such a device. A uniformly distributed flow of air is produced aroundthe nozzle stem of the device. An ultrasonically produced spray at thetip of the nozzle stem is immediately entrained in the air stream. Anadjustable focusing mechanism on the air shroud allows complete controlof the width of the spray. The spray envelope is bow-shaped. The widthof the bow is controlled by moving the focus adjust mechanism in andout. The distance between nozzle and substrate can be varied from nearcontact to approximately two inches. Liquid may be introduced onto anatomizing surface through a large, non-clogging feed channel running thelength of the nozzle. Liquid absorbs the vibrational energy resulting inthe atomization of the liquid. One such ultrasonic fluid ejection deviceis that manufactured by Sono-Tek Corporation, Milton N.Y., and soldunder the trademark AccuMist™.

[0069] The fluid that is delivered in accordance with the presentinvention is usually a liquid, the nature of which depends on the natureof the reagents to be dispensed to the surface. The liquid may be aprotic solvent or an aprotic solvent. Usually, it is desirable toconduct reactions in a protic solvent because of the nature of thereagents and surfaces involved. The liquid may be an aqueous medium thatis solely water or solely an organic solvent. An aqueous medium maycontain from about 0.01 to about 97 or more volume percent of acosolvent such as an organic solvent. Organic solvents include by way ofillustration and not limitation oxygenated organic solvents of from 1-6,more usually from 1-4, carbon atoms, including alcohols such asmethanol, ethanol, propanol, etc., ethers such as tetrahydrofuran, ethylether, propyl ether, etc., dimethylformamide, dimethylsulfoxide,1,4-dioxane, N-methyl-2-pyrrolidone (NMP), acetonitrile and the like.Usually these cosolvents, if used, are present in less than about 70weight percent, more usually in less than about 30 weight percent.

[0070] The pH for the liquid depends on the nature of the reagents,i.e., deprotection, protection, wash solution and the like and isgenerally selected to achieve optimum reaction between the molecules andreagents or to effectively wash a surface. The pH range is broad becausesome of the reagents dispensed include acids and bases. The pH isusually in the range of about 1 to about 14. Among the factors that mustbe considered are the pH dependence of the reactive molecules, thestability of the molecules at different pH values, and so forth. Variousbuffers may be used to achieve the desired pH and maintain the pH duringthe reaction. Illustrative buffers include acetate, borate, phosphate,carbonate, and the like. The particular buffer employed is not criticalto this invention as long as the buffer does not react unintentionallywith the reagents or the synthesized molecules. Further, in anindividual reaction or a wash step, one or another buffer may bepreferred.

[0071] The fluid ejection device is in fluid communication with areservoir containing the liquid to be dispensed. It is within thepurview of the present invention that the fluid ejection device is inalternating fluid communication with a plurality of reservoirs eachcontaining a different liquid. The liquids may differ by the presence ofdifferent reagents and the like. The number of steps for dispensingliquids in accordance with the present invention depends on the natureof the chemical reaction such as synthesis or diagnostic procedure,being conducted, e.g., in situ synthesis, synthesis by directattachment, assay for an analyte, etc. The number of separate distinctsteps for dispensing liquids may be as few as one or a great as five forany particular reagent dispensed. Furthermore, for any one of theseparate distinct steps liquid may be applied by dispensing such liquidone or more times. As mentioned above, one of the advantages that may berealized with the present invention is that the liquid may be applied inthin layers and several applications of thin layers of liquid may beemployed for each reaction or wash and the like.

[0072] In one embodiment of the present invention phosphoramiditereagents are deposited in an array pattern on a substrate by means ofinkjet technology. The substrate surface is then coated with anoxidation reagent by applying the liquid oxidation reagent to thesurface using a dispense head activated by ultrasonic energy. Thesurface of the substrate is then washed with a wash solution such as anorganic solvent, e.g., acetonitrile, to remove excess and unreactedreagents. The wash liquid may be applied using a dispense head activatedby ultrasonic energy. The surface is coated uniformly with a deblockingreagent applied again using a dispense head activated by ultrasonicenergy. The surface is washed as described above and phosphoramiditereagents are applied in a pattern using inkjet technology as discussedabove. The steps described above are repeated for a sufficient number oftimes to create the desired length of oligonucleotides on the array.

[0073] The oligonucleotide arrays constructed in accordance with thepresent invention may be used to carry out nucleic acid hybridization ina diagnostic fashion. To this end the array is exposed to a solutioncontaining the polynucleotide analytes in the usual manner and labeledDNA fragments selectively hybridize at sites where a complementaryoligonucleotide is found. The present method may be employed to dispensethe solution containing the polynucleotide analytes, a wash solution,and so forth.

[0074] As mentioned above, one embodiment of the present invention, byway of illustration and not limitation, is an apparatus for forming anarray of polynucleotides at discrete sites on a surface. The apparatuscomprises a device for dispensing reagents to predetermined discretesites on said surface and a fluid ejection device activated by means ofultrasonic energy. The fluid ejection device dispenses a volume of aliquid as particles of uniform diameter to uniformly coat the surfacewith liquid. The reagents are selected from the group consisting ofnucleotides and polynucleotides. Referring to FIG. 2, apparatus 30 isshown that comprises first platform 32 and second platform 33, eachmounted on a main platform (not shown) of apparatus 30. Transfer robot34 is also mounted on the main platform of apparatus 30 and comprisesbase 34 a, arm 34 b that is movably mounted on base 34 a, and wafertransporter 34 c that is attached to arm 34 b.Substrate wafer 36 isremovably resting on first linear stage 39, which is movably mounted onfirst platform 32 and moveable in the x direction. Second linear stage38 is affixed to first platform 32. Inkjet piezo module 40 is mounted onsecond linear stage 38 and is movable in the y direction. Secondplatform 33 comprises third linear stage 41, which is mounted on secondplatform 33 and is moveable in the x-direction. Fourth linear stage 42is affixed to second platform 33 and ultrasonic fluid ejection device 44is mounted on fourth linear stage 42. Device 44 may be affixed centrallyon fourth linear stage 42 or it may be mounted on fourth linear stage 42to be moveable in the y-direction.

[0075] In use, substrate wafer 36, on which an array of polynucleotidesis to be formed, is removably secured on first linear stage 39. Inkjetpiezo module 40 is activated to deposit phosphoramidite reagents on thesurface of wafer 36 at discrete sites. First linear stage 39 is movedalong the x-axis and inkjet piezo module 40 is moved along the y-axis.Subsequently, transfer robot 34 is activated to move arm 34 b so thatwafer transporter 34 c removes substrate wafer 36 from first linearstage 39. Arm 34 b of transfer robot 34 is moved so that wafertransporter 34 c delivers substrate wafer 36 to third linear stage 41.Ultrasonic fluid ejection device 44 is activated to dispense a liquidreagent that comprises an oxidizing reagent to uniformly coat thesurface of wafer 36 with a thin layer of the liquid reagent. In thatregard third linear stage 41 is moved along the x-axis; and, if device44 is moveably mounted on fourth linear stage 42, device 44 is movedalong the y-axis.

[0076] Additional steps in the synthesis of an array of polynucleotideson the surface of wafer 36 are carried out as described above usingapparatus 30. For example, while substrate wafer 36 remains at secondplatform 33, ultrasonic fluid ejection device 44 is activated todispense a wash liquid. Prior to contact with a wash liquid, wafer 36may be treated to remove excess and unreacted reagents. Such treatmentmay be by spinning, suction or vacuum, contact with an inert gas and soforth. For example, an air jet may be mounted on fourth linear stage 42and may be used to dispense a stream of an inert gas such as nitrogen,argon and the like to dry the surface of wafer 36.

[0077] For dispensing wash liquid and other liquids, device 44 is influid communication with more than one reservoir (reservoirs not shown)containing the appropriate liquid reagents or wash liquids for use inthe above steps or in subsequent steps. Suitable valving mechanisms areemployed to permit establishment and disengagement of various desiredfluid communications.

[0078] Following the washing step and while substrate wafer 36 remainsat second platform 33, ultrasonic fluid ejection device 44 is activatedto dispense a deblocking reagent to uniformly coat the surface ofsubstrate wafer 36. Again, excess liquid and reagents may be removedfrom the surface of wafer 36 and, while substrate wafer 36 remains atsecond platform 33, ultrasonic fluid ejection device 44 is activated todispense a wash liquid to wash the surface of wafer 36. Subsequently,transfer robot 34 is activated to move arm 34 b so that wafertransporter 34 c removes substrate wafer 36 from third linear stage 41.Arm 34 b of transfer robot 34 is moved so that wafer transporter 34 cdelivers substrate wafer 36 to first linear stage 39. Inkjet piezomodule 40 is activated to deposit phosphoramidite reagents on thesurface of wafer 36 at discrete sites. It should be understood that theabove steps may be repeated a sufficient number of times so that thedesired polynucleotides are synthesized in an array on the surface ofsubstrate wafer 36. It should also be understood that each step mayinclude one or more dispensing actions in accordance with the presentinvention as explained hereinabove.

[0079] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0080] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

What is claimed is:
 1. A method for forming an array of polynucleotidesat discrete sites on a surface, said method comprising: (a) applyingreagents to predetermined discrete sites on said surface, said reagentsbeing selected from the group consisting of nucleotides andpolynucleotides, (b) dispensing a volume of a liquid to uniformly coatsaid surface with liquid, said volume of liquid being dispensed asparticles of uniform diameter through a fluid ejection device activatedby means of ultrasonic energy, and (c) optionally repeating step (a) orstep (b).
 2. A method according to claim 1 wherein said liquid comprisesan agent selected from the group consisting of washing solutions,buffers, deblocking agents, blocking agents, deprotection agents,protection agents and phosphoramidite reagents.
 3. A method according toclaim 1 wherein said reagents are nucleotides and steps (a) and (b) arerepeated for a number of times sufficient to form said array ofpolynucleotides.
 4. A method according to claim 3 whereinphosphoramidite chemistry is employed.
 5. A method according to claim 1wherein said surface is essentially planar.
 6. A method according toclaim 1 wherein said liquid is dispensed from an outlet of said fluidejection device as a mist.
 7. A method according to claim 1 wherein step(a) is carried out by an application technology selected from the groupconsisting of printing technology, masking technology, ultrasonictechnology and combinations thereof.
 8. A method according to claim 1wherein said uniform particles have a diameter on the average of about10 microns to about 150 microns.
 9. A method according to claim 1wherein said ultrasonic energy has a frequency of about 5 kilohertz toabout 300 kilohertz.
 10. A method according to claim 1 wherein saidvolume of fluid is about 1 nanoliter to about 1000 nanoliters per squarecentimeter.
 11. A method according to claim 1 wherein said surface ismounted on a linear stage and moved in position relative to said fluidejection device above said stage.
 12. A method according to claim 1wherein said surface is rotated relative to said fluid ejection deviceand said fluid ejection device is moved radially relative to saidsurface.
 13. A method according to claim 1 wherein said fluid ejectiondevice is in fluid communication with a reservoir containing saidliquid.
 14. A method according to claim 1 wherein said fluid ejectiondevice is in alternating fluid communication with a plurality ofreservoirs each containing a different liquid.
 15. A method for formingan array of polynucleotides at discrete sites on a surface, said methodcomprising: (a) applying nucleotide reagents to predetermined discretesites on said surface, (b) dispensing a volume of a liquid of about 1nanoliter to about 1000 nanoliters per square centimeter to uniformlycoat said surface with liquid, said volume of liquid being dispensed asparticles of uniform diameter of about 10 microns to about 150 micronsthrough a fluid ejection device activated by means of ultrasonic energyat a frequency of about 5 kilohertz to about 300 kilohertz, and (c)optionally repeating step (a) or step (b).
 16. A method according toclaim 15 wherein said liquids comprise agents selected from the groupconsisting of washing solutions, buffers, deblocking agents, blockingagents, deprotection agents and protection agents.
 17. A methodaccording to claim 15 wherein step (b) is repeated at least one timeprior to repeating step (a) to dispense multiple layers of said liquidon said surface.
 18. A method according to claim 17 wherein said liquidis dispensed as a layer of about 5 to 100 microns in thickness.
 19. Aapparatus for forming an array of polynucleotides at discrete sites on asurface, said apparatus comprising: (a) a device for dispensing reagentsto predetermined discrete sites on said surface, said reagents beingselected from the group consisting of nucleotides and polynucleotidesand (b) a fluid ejection device activated by means of ultrasonic energyfor dispensing a volume of a liquid as particles of uniform diameter touniformly coat said surface with liquid.
 20. An apparatus according toclaim 19 wherein said device for dispensing reagents is an inkjetdevice.